There are two broad goals of the proposal. The first is to contribute to the development of biophysical imaging technologies for use in Saccharomyces cerevisiae, which has traditionally been a model system for genetic and biochemical studies but has seen fewer applications of advanced biophysical techniques, with no publications in budding yeast implementing fluorescence correlation spectroscopy (PCS) or spectral imaging. Initial results show these techniques work well with budding yeast. The combination of the powerful genetic advantages of working with yeast with application of emerging biophysical techniques would raise the bar for the potential of interdisciplinary studies. It is the second goal of this proposal to outline biophysical experiments that will shed light on molecular interactions involved in the secretory processes in-vivo. To examine exocytosis, we propose to examine the role of myosin-mediated transport in establishment and maintenance of the asymmetrical molecular distribution required for cellular growth (Aim1). Myosin transport occurs on actin cables, which reside in the cytosol. Fluorescence signal is far too dim to observe with conventional imaging, yet PCS is able to examine dynamics with dim fluorescence signal. Preliminary results outlined in the proposal demonstrate the capability of PCS to resolve myosin motion in the cytosol. Arp2/3 mediated actin polymerization is hypothesized to play an important role in formation and scission of endocytic vesicles, believed to occur in actin patches. Although imaging studies have detailed the arrival of endocytic signaling and Arp2/3 activating proteins to actin patches, it is not certain which of these proteins activates Arp2/3 actin polymerization in-vivo. Aim 2 will examine Arp2/3 activation in solution with time- resolved FRET to further the understanding of the conformational changes that occur to Arp2/3 upon binding of activators. Based on the results of Aim 2, a FRET biosensor for Arp2/3 activation will be developed and applied in-vivo to determine when and how Arp2/3 is activated using spectral imaging (Aim 3). The coordination of Arp2/3 activation and endocytic vesicle internalization will be detailed for the first time. Relevance to public health: Cell polarization is a necessary precursor for processes such as cell motility and division. Myosin-mediated transport and endocytic recycling play major roles in establishing and maintain the asymmetric protein distribution required for polarization. Understanding the secretory processes and Arp2/3 activation has relevance to the actin cytoskeleton, cell division and cell motility, and thus to cancer growth and metastasis. [unreadable] [unreadable] [unreadable]